Peptide with anti-obesity and anti-diabetes activity and use thereof

ABSTRACT

A peptide and a peptide complex of the present invention exhibit an anti-obesity effect by inhibiting fat accumulation and decomposing already accumulated fat, and exhibit an excellent effect with respect to diabetes by effectively reducing blood sugar. The peptide and the peptide complex of the present invention decrease the expression of PPARγ, ACC, and aP2, which are adipogenic markers, increase the expression of pHSL, AMPK-α1, CGI-58, and ATGL, which are lipolytic factors, and reduce the size of fat cells and blood cholesterol values. The peptide and the peptide complex of the present invention, which have excellent activity and safety, can be advantageously applied to drugs and quasi-drugs.

TECHNICAL FIELD

This application claims priority to Korean Patent ApplicationNo.10-2015-0059648, filed Apr. 28, 2015, to the KIPO. The content of theabove applications are incorporated by reference in their entirety.

The present invention relates to a peptide with anti-obesity andanti-diabetes activity, and use thereof.

BACKGROUND ART

In Korea, dietary fat intake has recently increased with the growth ofeconomy and the westernization of diet life, and onset of metabolicdiseases such as obesity, diabetes, hyperlipidemia, hypertension,arteriosclerosis, and fatty liver increased due to insufficientexercise. In addition, obesity is an aesthetic problem to people whogenerally tend to prefer to slim body types as well as being associatedwith various disorders.

To date, therapeutic agents for obesity may be largely divided intodrugs that act on the central nervous system to affect appetite anddrugs that act on the gastrointestinal tract to inhibit uptake. Drugsacting on the central nervous system were placed on the market asanti-obesity drugs which work on the serotonin (5-HT) in the nervoussystem such as fenfluramine, dexfenfluramine and the like, on thenoradrenaline nervous system such as ephedrine and caffeine, and on boththe serotonin and the noradrenaline nervous system such as recentlydeveloped sibutramine, as classified by acting mechanisms.Representative of anti-obesity drugs acting on the gastrointestinaltract is orlistat, approved as a therapeutic agent for obesity, whichinhibits intestinal lipase to reduce fat uptake. There are problems withsome of the pre-existing drugs. For example, fenfluramine and the likehave been prohibited from being marketed due to the side effect ofincurring primary pulmonary hypertension or valvular heart disease, andother drugs cannot be applied to patients with heart failure or kidneyfailure due to the occurrence of blood pressure reduction or lacticacidosis.

Diabetes is a group of metabolic disorders caused when insulin isinsufficiently secreted or in does that do not enable normal function(DeFronzo, 1988) and is characterized by hyperglycemia, that is, highblood sugar levels over a prolonged period, which causes varioussymptoms and syndromes, with glucose in urine. In recent years, theprevalence of obesity, particularly, abdominal obesity has increased,leading to the explosion of the prevalence of diabetes.

As of 2000, diabetes patients were estimated to be 170 million worldwideand expected to increase to 370 million people in 2030. However, a 2008analysis report showed that the number of diabetes patients may havealready reached 350 million worldwide (Danaei et al., 2011), with farmore significant aggregation than expectation. It is reported that morethan about 80% of type 1 diabetes patients are obese whereas only lessthan 10% of (non-)obese patients have diabetes (Harris et al. 1987). Thecorrelation between diabetes and obesity is attributed to the fact thatadipokines and free fatty acids are irregularly secreted to induce fattyacids to accumulate in insulin-sensitive tissues such as beta cells,kidneys, liver, heart, etc., resulting in lipotoxicity. If left withoutsuitable treatment, chronic hyperglycemia may be prone to incurringvarious pathological symptoms including retinopathy, renal dysfunction,neuropathy, and vascular disorder. Indispensable for preventing suchcomplications is effective blood sugar management.

Nowadays, the control of blood sugar levels is accomplished by lifestyleimprovement (diet therapy, exercise therapy), and medications. However,diet therapy or exercise therapy is difficult to strictly manage andpractice, with limitations of the effects thereof. Hence, most patientswith diabetes rely on the control of blood sugar levels by medicationssuch as insulin, insulin secretagogues, insulin sensitizer, andhypoglycemic agents, as well as lifestyle improvement.

Insulin produced using a recombinant method is used as a drugindispensable to type 1 diabetes patients and type 2 diabetes patientswhich fail to control blood sugar levels, and is advantageous in bloodsugar control. However, it suffers from the disadvantage of repulsion tosyringe needles, difficulty in administration, hypoglycemic risk, andweight gain.

Meglitinides, a kinds of insulin secretagogues, are short-acting agentsand are taken before meals. Among them are NovoNorm (repaglinide),Fastic (nateglinide), and Glufast (mitiglinide). Insulin sensitizers arecharacterized by almost no hyperglycemic incurrence when taken alone,and may be exemplified by biguanide drugs, such as metformin, andthiazolidinedione drugs such as Avanida (rosiglitazone) and Actos(pioglitazone).

Recently, GLP-1 agonists have been developed using the action ofglucagon-like peptide-1, which is an insulin secretion-stimulatinghormone, and include exenatide and Victoza (liraglutide). In addition,DDP-4 inhibitors, which inhibit the action of DPP (dipeptidylpeptidase-4), an enzyme responsible for the rapid inactivation of GLP-1,are newly developed drugs and are representatively exemplified byJanuvia (ingredient name: sitagliptin). However, those drugs arereported to have side effects of hepatoxicity, gastrointestinaldisorders, cardiovascular disorders, and carcinogenicity. Anotherproblem with the drugs is a high annual treatment cost, which is abarrier to the treatment of diabetes. Indeed, health care costs ofpre-diabetes and diabetes approached about 200 trillion won in the USAas of 2007 (Dall et al., 2010), and health care costs of obesity arealso near 150 trillion won only in the USA as of 2008 (Finkelstein etal., 2009). Therefore there is an urgent need for the development of adrug that can effectively lower blood glucose levels and can be appliedto both diabetes and obesity-induced diabetes, with less side effects.

For this, the present inventors have recently paid attention to energymetabolism-regulating mechanisms in order to find an improved method forthe treatment of obesity, and have made research of signals responsiblefor lipid accumulation and proteins affecting lipid accumulation uponthe intake of high-fat diets in humans, with the premise that thecompound to be developed should of higher safety (lower toxicity). As aresult of research on signals for suppressing the expression of proteinsresponsible for fat accumulation and for degrading accumulated fat andon proteins involved in the signaling, the present inventors succeededin developing peptides that promote lipolysis. In addition, the peptidesof the present invention exhibit outstanding therapeutic efficacy ondiabetes and obesity-induced diabetes. The fat accumulation induced byhigh-fat diets, the suppression of insulin signaling attributed to fataccumulation in the liver or muscle, and resulting insulin tolerance arecauses of diabetes. Each and complexes of the peptides according to thepresent invention are therapeutically effective for such diabetes andobesity-induced diabetes.

Throughout this specification, reference is made to many papers andpatent documents, with citations thereof indicated. The disclosures ofthe cited papers and patent documents are herein entirely incorporatedby reference and thus the level of technical field to which the presentinvention belong and contents of the present invention are explainedmore definitely.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

Culminating in the present invention, intensive and thorough research onthe development of plural outstanding peptides having biologicallyeffective activity, conducted by the present inventors, led to thefinding that peptides having the amino acid sequences of SEQ ID NOS: 1to 7 exhibit not only anti-obesity effects by suppressing high-fatdiet-induced fat accumulation and degrading already accumulated fat, butalso high therapeutic effects on diabetes and obesity-induced diabetes,and diabetes complications.

Accordingly, an object of the present invention is to provide peptideshaving the amino acid sequences of SEQ ID NOS: 1 to 7.

Another object of the present invention is to provide a peptide havinganti-obesity or anti-diabetes activity.

A further object of the present invention is to provide a peptidecomplex having anti-obesity or anti-diabetes activity

A still further object of the present invention is to provide apharmaceutical composition for the prevention or treatment of obesity.

Still another object of the present invention to provide apharmaceutical composition for the prevention or treatment of diabetes.

Other purposes and advantages of the present invention will becomeclarified by the following detailed description of the invention,claims, and drawings.

Technical Solution

One embodiment of the present invention provides a peptide having oneselected from the group consisting of the amino acid sequences of SEQ IDNOS: 1 to 7.

Another embodiment of the present invention provides a peptide ofanti-obesity and anti-diabetes activity having one selected from thegroup consisting of the amino acid sequences of SEQ ID NOS: 1 to 7.

Provided according to another embodiment of the present invention is apeptide complex of anti-obesity and anti-diabetes activity, composed ofthe following peptide combination:

(a) a peptide having the amino acid sequence of SEQ ID NO: 1;

(b) a peptide having the amino acid sequence of SEQ ID NO: 2 or 3; and

(c) a peptide having the amino acid sequence of SEQ ID NO: 6 or 7.

As a result of the effort of the present inventors to develop pluraloutstanding peptides having biologically effective activity, it wasfound that peptides having the amino acid sequences of SEQ ID NOS: 1 to7 suppress high-fat diet-induced fat accumulation and degrade alreadyaccumulated fat, thus exhibiting an anti-obesity effect and atherapeutic effect on diabetes and obesity-induced diabetes, or diabetescomplications.

As used herein, the term “peptide” refers to a linear molecule of aminoacid residues linked by peptide bonds. The peptides of the presentinvention may be prepared using chemical synthesis methods known in theart, especially solid-phase synthesis techniques (Merrifield, J. Amer.Chem. Soc. 85:2149-54(1963); Stewart, et al., Solid Phase PeptideSynthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111(1984)) or aliquid-phase synthesis method (U.S. Pat. No. 5,516,891).

In order to select regions of the amino acid sequences thereof andincrease the activity thereof, the peptides of the present invention maybe modified at N- or C-terminals thereof. Through such modification, thepeptides of the present invention may be imparted with a prolongedhalf-life after in vivo administration.

Further, C-terminals of the peptides of the present invention may bemodified with a hydorxy group (—OH), an amino group (—NH2), an azidegroup (—NHNH2), etc. while N-terminals may be coupled with a protectingradical consisting of the group consisting of acetyl, fluorenyl methoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, and polyethylene glycol(PEG).

Through the above stated amino acid modification, the peptides of thepresent invention can greatly increase in stability. As used herein, theterm “stability” is intended to refer to both in vivo stability andstorage stability (e.g., stability during storage at room temperature).The protecting group acts to protect the peptides of the presentinvention against the attack of proteinases in vivo.

According to one embodiment of the present invention, the peptides ofthe present invention exhibit the effect of suppressing high-fatdiet-induced fat accumulation and degrading already accumulated fat,decrease the expression of the adipogenic markers PPARγ, ACC, and aP2,increase the expression of the lipolytic factors pHSL, AMPK-α1, CGI-58,and ATGL, reduce the size of adipose cells, and lower blood cholesterollevels. These results indicate that the peptides of the presentinvention have excellent therapeutic effects on obesity, diabetes, andobesity-induced diabetes.

Not only individual peptides of SEQ ID NOS: 1 to 7, but also a complexthereof exhibits excellent anti-obesity and anti-diabetes activity.

According to the present invention, the peptides of SEQ ID NOS: 3, 5,and 7 correspond respectively to those of SEQ ID NOS: 2, 4, and 6, withthe exception that the Cys residue is substituted with the Ser residue.The corresponding paired peptides are almost identical in terms ofanti-obesity and anti-diabetes activity.

In accordance with an embodiment of the present invention, the peptidecomplex exhibiting anti-obesity or anti-diabetes activity is composed ofa peptide having the amino acid sequence of SEQ ID NO: 1; a peptidehaving the amino acid sequence of SEQ ID NO: 2 or 3; and a peptidehaving the amino acid sequence of SEQ ID NO: 6 or 7.

According to another embodiment of the present invention, the peptidecomplex of the present invention is composed of peptides having therespectively amino acid sequences of SEQ ID NOS: 1, 3, and 7.

Contemplated in accordance with another aspect of the present inventionis a pharmaceutical composition comprising the peptide or peptidecomplex of the present invention as an effective ingredient forpreparing or treating obesity.

Superior in terms of anti-adipogenetic and lipolytic functions, thepeptide or peptide complex of the present invention can be useful forthe prophylaxis or therapy of obesity.

A further aspect of the present invention provides a pharmaceuticalcomposition comprising the peptide or peptide complex of the presentinvention as an effective ingredient for preventing or treatingdiabetes.

Functioning to effectively lower an increased blood sugar level indiabetes animal models, the peptide or peptide complex of the presentinvention can find applications in the prophylaxis or therapy ofdiabetes.

According to some particular embodiments of the present invention, thecomposition of the present invention is a pharmaceutical compositioncomprising: (a) a pharmaceutically effective amount of the peptide orpeptide complex of the present invention; and a pharmaceuticallyacceptable carrier.

The term “pharmaceutically effective amount”, as used herein, means asufficient amount to achieve the above-stated efficacy or activity ofthe peptide.

The pharmaceutically acceptable carrier contained in the pharmaceuticalcomposition of the present invention may be that commonly used in drugformulations and include, but are not limited to, lactose, dextrose,sucrose, sorbitol, mannitol, starch, acacia gum, calcium carbonate,alginate, gelatin, calcium silicate, microcrystalline cellulose,polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, andmineral oil. In addition to those ingredients, the pharmaceuticalcomposition of the present invention may further comprise a lubricant, ahumectant, a sweetener, a flavorant, an emulsifier, a suspending agent,and a preservative. With regard to pharmaceutically acceptable carriersand agents suitable for use, reference may be made to Remington'sPharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may beadministered orally or parenterally. For parenteral administration,intramuscular, intravenous, subcutaneous, intraperitoneal, topical, ortranscutaneous routes may be used.

The dosage of the pharmaceutical composition according to the presentinvention may vary depending on various factors, including dosage form,administration modality, the patient's age, weight, gender, state ofhealth, diet, the time of administration, the route of administration,excretion rate, sensitivity, etc. For example, the pharmaceuticalcomposition according to the present invention may be administered at adaily dose in the range of 0.0001 to 1,000 μg.

The pharmaceutical composition according to the present invention may beprepared in single-dose forms or in multi-dose packages using apharmaceutically acceptable carrier and/or excipient according to amethod that may be easily carried out by those skilled in the art.Herein, the formulation of the pharmaceutical composition may be asolution, suspension or emulsion of the pharmaceutical composition inoil or aqueous medium, or an extract, powder, granule, tablet or capsulecontaining the pharmaceutical composition, and may further comprise adispersing agent or a stabilizer.

Advantageous Effects

Features and advantages of the present invention are summarized asfollows:

(i) the peptides and the peptide complex of the present inventionexhibit not only an anti-obesity effect by suppressing fat accumulationand degrading already accumulated fats, but also an outstandingtherapeutic effect on diabetes by effectively reducing blood sugarlevels.

(ii) the peptides and the peptide complex of the present inventiondecrease the expression of the adipogenic markers PPARγ, ACC, and aP2,increase the expression of the lipolytic factors pHSL, AMPK-60 1,CGI-58, and ATGL, thus reducing adipocyte sizes and blood cholesterollevels.

(iii) the peptides and the peptide complex of the present invention haveexcellent activity and safety and thus can be advantageously applied todrugs and quasi-drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows lipids accumulated after treatment with peptides of thepresent invention, as analyzed by Oil red O staining: (a) peptide of SEQID NO: 1, (b) peptide of SEQ ID NO: 3, and (c) peptide of SEQ ID NO: 5.

FIG. 2 shows results of lipid accumulation after treatment with thepeptide complex of the present invention, as analyzed by Oil red Ostaining.

FIG. 3 shows measurement results of the expression levels of the geneaP2, which is involved in adipogenesis, after treatment with thepeptides of the present invention: (a) peptide of SEQ ID NO: 1, (b)peptide of SEQ ID NO: 3, and (c) peptide of SEQ ID NO: 5.

FIG. 4 shows measurement results of expression levels of PPARγ, ACC, andaP2 genes, which play an important role in adiapgenesis, after treatmentwith various concentrations of the peptide complex of the presentinvention.

FIG. 5 shows measurement results of expression levels of PPARγ andphospho-HSL, which play an important role in adipogenesis, after variousconcentrations of the peptide complex of the present invention.

FIG. 6 shows measurement results of expression levels of AMPK-α1 andCGI58 genes, which are involved in the degradation of accumulated fats,after treatment with the peptides and peptide complex of the presentinvention: (a) peptide of SEQ ID NO: 1, (b) peptide of SEQ ID NO: 3, (c)peptide of SEQ ID NO: 5, and (d) a complex of peptides of SEQ ID NOS: 1,3, and 7.

FIG. 7 shows measurement results of ATGL, a protein involved in thedegradation of accumulated fats, after treatment with variousconcentrations of the peptide complex of the present invention.

FIG. 8 shows results of expression levels of the Phospho-HSL proteininvolved in the degradation of accumulated fats, after treatment withthe peptides of the present invention: (a) peptide of SEQ ID NO: 1, (b)peptide of SEQ ID NO: 3, (c) peptide of SEQ ID NO: 5, and (d) a complexof peptides of SEQ ID NOS: 1, 3, and 7, as measured by immunostaining.

FIG. 9 shows measurement results of glycerol produced after treatmentwith various concentrations of the peptides complex of the presentinvention.

FIG. 10a shows adipose tissues degraded in obese mouse experiment modelsafter treatment with the peptide complex of the present invention. FIG.10b shows sizes and numbers of the adipose tissues degraded in obesemouse experiment models after treatment with the peptide complex of thepresent invention.

FIG. 11 shows results of the expression levels of the Phospho-HSLprotein which is involved in the degradation of accumulated fats aftertreatment with the peptide complex of the present invention, as measuredby immunostaining.

FIG. 12 shows changes in body weight (g) and diet intake of obese miceafter treatment with the peptide complex of the present invention.

FIG. 13 shows images of obese mice after treatment of the peptidecomplex of the present invention.

FIG. 14 shows results of fat distribution in obese mouse models inducedby feeding a high-fat diet to the experimental animal model C57BL/6, asanalyzed by micro-CT.

FIG. 15 shows images of the adipocyte tissues extracted from obese mousemodels induced by feeding a high-fat diet to the experimental animalmodel C57BL/6, after treatment with the peptide complex of the presentinvention.

FIG. 16a shows morphological images of the adipocytes in adipose tissuestaken from obese mouse models induced by feeding a high-fat diet to theexperimental animal model C57BL/6, after treatment with the peptidecomplex of the present invention. FIG. 16b shows size results of theadipocytes in the adipose tissues taken from obese mouse models inducedby feeding a high-fat diet to the experimental animal model C57BL/6,after treatment with the peptide complex of the present invention.

FIG. 17 shows measurement results of the expression levels of thephosphor-HSL protein, which is involved in lipolysis, in adipocytes ofadipose tissues taken from obese mouse models induced by feeding ahigh-fat diet to the experimental animal model C57BL/6, after treatmentwith the peptide complex of the present invention.

FIG. 18 shows measurement results of cholesterol levels in blood samplestaken from obese mouse models induced by feeding a high-fat diet to theexperimental animal model C57BL/6, after treatment with the peptidecomplex of the present invention.

FIG. 19 shows measurement results of glucose levels in blood samplestaken from obese mouse models induced by feeding a high-fat diet to theexperimental animal model C57BL/6, after treatment with the peptidecomplex of the present invention.

FIG. 20 shows changes in blood sugar level in obesity-induced DB/DBmouse models after treatment with the peptide complex of the presentinvention.

FIG. 21 shows changes in blood cholesterol level in obesity-inducedDB/DB mouse models after treatment with the peptide complex of thepresent invention.

FIG. 22 shows changes in blood sugar level in obesity-induced DB/DBmouse models after treatment with the peptides of the present invention:(a) peptide of SEQ ID NO: 1, (b) peptide of SEQ ID NO: 3, and (c)peptide of SEQ ID NO: 5.

FIG. 23 shows measurement results of expression levels of IGF-1 andinsulin after treatment with the peptide of SEQ ID NO: 7.

FIG. 24 shows changes in blood sugar level in obesity-induced DB/Dbmouse model after treatment with the peptide of SEQ ID NO: 7.

FIGS. 25a-25d show changes in blood sugar levels in diabetes patientshaving high blood glucose levels after treatment with the peptidecomplex of the present invention.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in further detailwith reference to examples. It will be obvious to a person havingordinary skill in the art that these examples are illustrative purposesonly and are not to be construed to limit the scope of the presentinvention. Thus, the substantial scope of the present invention will bedefined by the appended claims and equivalents thereof.

EXAMPLES Synthesis Example 1: Peptide Synthesis

In a reactor, 700 mg of chlorotrityl chloride resins (CTL resins, Novabiochem Cat No. 01-64-0021) was added with 10 ml of methylene chloride(MC) and stirred for 3 min. After removal of the solvent, 10 ml ofdimethyl formamide (DMF) was added. The solution was stirred again for 3min, and then the solvent was removed. To the reactor was added 10 ml ofa dichloromethane solution, followed by 200 mmole of Fmoc-Asn(Trt)-OH(Bachem, Swiss) and 400 mmole of diisopropyl ethylamine (DIEA). Thereactants were well dissolved and reacted while stirring for 1 hour.Thereafter, the solution was washed, and reacted with a solution ofmethanol and DIEA (2:1) in DCM (dichloromethane) for 10 min. Subsequentto washing with an excess of DCM/DMF (1:1), the solvent was removed.Then, 10 ml of dimethyl formamide (DMF) was added, followed by stirringfor 3 min. After removal of the solvent, 10 ml of a deprotectingsolution (20% piperidine/DMF) was added to the reactor. Stirring at roomtemperature for 10 min was precedent to the removal of the solvent. Thedeprotecting solution was added in the same amount and then removedafter 10 min of reaction. Washing was performed twice with DMF, oncewith MC, and once with DMG for 3 min each wash to afford Asn-CTL resins.In another reactor, 200 mmole of Fmoc-Arg(Pbf)-OH(Bachem, Swiss), 200mmole of HoBt, and 200 mmole of Bop were added to 10 ml of a DMFsolution and well dissolved by stirring. To the reactor, 400 mmole ofDIEA was added in two aliquots, followed by stirring for at least 5 minto the complete dissolution of the solid. The dissolved amino acidmixture solution was introduced into the reactor in which thedeprotected resins were placed, followed by stirring for 1 hour at roomtemperature for reaction. After the reaction liquid was removed,stirring was carried three times for 3 min each time, together with aDMF solution which was then removed. A small amount of the reactionresins was taken and used in a Kaiser test (ninhydrin test) forexamining an extent of the reaction. The same deprotection reaction wasperformed twice with the deprotecting solution to give Arg-Asn-CTLresins. The resins were sufficiently washed with DMF and MC before anadditional Kaiser test. The following amino acid attachment experimentswere carried out in the same manner as described above. According toselected amino acid sequences, reactions were sequentially induced withFmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Leu-OH in that order. TheFmoc-protecting group was removed by reacting twice with a deprotectingsolution for 10 min for each reaction and then well washing. Aceticanhydride, DIEA, and HoBt were added and subjected to acetylation for 1hour. The peptidyl resins thus obtained were washed with DMF, MC, andmethanol three times each. The resins were dried with nitrogen gasslowly flowed and then were completely vacuum-dried under a P2O5atmosphere. The resins were reacted for 2 hours at room temperature with30 ml of a leaving solution (trifluoroacetic acid 81.5%, distilled water5%, thioanisole 5%, phenol 5%, EDT 2.5%, and TIS 1%) whileintermittently agitating. The resins were filtered and washed with asmall volume of TFA solution, after which the filtrate was combined withthe mother liquid. After distillation at a reduced pressure to reducethe total volume by two, 50 ml of cold ether was used to induceprecipitation, and the precipitates thus formed were collected bycentrifugation and washed twice with cold ether. After removal of themother liquid, the remainder was sufficiently dried under a nitrogenatmosphere to afford 0.85 g of the unpurified peptide of SEQ ID NO: 1NH2-Leu-Lys-Thr-Arg-Asn-COOH (yield: 92%). Synthesis was made ofNH2-Lys-Gly-Ala-Cys(Ser)-Thr-Gly-Trp-Met-Ala-COOH in an amount of 0.78 gas peptides of SEQ ID NOS: 2 and 3 (yield: 82%),NH2-Ala-Cys(Ser)Thr-Leu-Pro-His-Pro-Trp-Phe-Cys(Ser)-COOH in an amountof 0.92 g as peptides of SEQ ID NOS: 4 and 5 (yield: 85%), andNH2-Cys(Ser)-Asp-Leu-Arg-Arg-Leu-Glu-Met-Tyr-Cys(Ser)-COOH in an amountof 0.76 g as peptides of SEQ ID NOS: 6 and 7 (yield: 88%). The peptidesof SEQ ID NOS: 1, 2, 4, and 6 were found to have molecular weights of630.7 (calc.: 630.7), 924.5 (calc.: 924.1), 1236 (calc.: 1236.5), and1301.5 (calc.: 1301.5), respectively, as measured by mass spectrometry.

TABLE 1 Analysis Amino Acid (Mass spectrometry) Peptide SequenceMeasured Calculated SEQ ID NO: 1 630.7 630.7 SEQ ID NO: 2 KGACTGWMA924.5 924.1 SEQ ID NO: 3 KGASTGWMA (908.0) SEQ ID NO: 4 ACYLPHPWFC 12361236.5 SEQ ID NO: 5 ASYLPHPWFS (1269.4) SEQ ID NO: 6 CDLRRLEMYC 1301.51301.5 SEQ ID NO: 7 SDLRRLEMYS

Meanwhile, peptides of SEQ ID NOS: 1, 3, and 7 were mixed in equalamounts to give a peptide complex which was evaluated for efficacy.

Example 1: Assay for Inhibitory Activity Against Adipogenesis 1-1. Assayfor Suppression of Lipid Accumulation by Use of Pre-Adipocyte (Oil Red OStaining)

The pre-adipocytes 3T3-L1 cells were grown to confluence and thenincubated for two days with various concentrations of the peptides in adifferentiation medium containing 10 μg/ml insulin, 0.1 μMdexamethasone, and 0.5 μM IBMX. The medium was exchanged every two dayswith a medium containing 10 μg/ml insulin. After differentiation wasinduced for 10 days, the generation of droplet in the cells was examinedby Oil Red O staining. The prepared 3T3-L1 adipocytes were washed withPBS, fixed with 3.7% formalin for one hour, and washed with 60%isopropanol. The resulting cells were dyed with Oil Red O reagent atroom temperature for 20 min. After removal of the Oil Red O reagent, thecells were washed three times with distilled water, and observed under aphase-contrast microscope. For quantitative analysis, fats wereextracted from the cells using 100% isopropanol, and the cells weretransferred in an amount of 200 μl/well into 96-well plates and measuredfor optical density at 500 nm using an ELISA reader.

Experimental data showed that treatment with peptides of SEQ ID NOS: 1,3, and 5 decreased extents of fat accumulation in cells, as measured byOil red O staining (FIGS. 1a-c ).

An extent of lipid accumulation in cells was also reduced when a complexof peptides of SEQ ID NOS: 1, 3, and 7 was applied by concentrations(FIG. 2).

1-2. Suppression of Expression of Genes Involved in Adipogenesis

3T3-L1 cells (pre-adipocytes) were seeded at a density of 3×105cells/well into 6-well plates. After 24 hours of culturing, the cellswere incubated at with predetermined concentrations (0.1, 1, and 10μg/ml) of the peptides for 14 days in a 37° C. incubator. Thereafter,the cells were harvested and treated with an RNA extraction solution(Easy Blue, Intron) to prepare RNA from which cDNA was then synthesizedusing an RT premix (Intron). PCR was performed using primers forantigenic markers (PPARγ, ACC, and aP2), and a PCR premix (Intron).

Target-specific primer sequences for PCR of adipogenic markers were asfollows: PPARγ forward primer sequence, 5′-TTTTCAAGGGTGCCAGTTTC-3′ andPPARγ reverse primer, 5′-AATCCTTGGCCCTCTGAGAT-3′ (annealing temperature,60° C.); ACC forward primer sequence, 5′-ACCTTACTGCCATCCCATGTGCTA-3′ andACC reverse primer, 5′-GTGCCTGATGATCGCACGAACAAA-3′ (annealingtemperature, 60° C.); aP2 forward primer sequence,5′-CATCAGCGTAAATGGGGATT-3′ and aP2 reverse primer,5′-ACACATTCCACCACCAGCTT-3′ (annealing temperature, 60° C.).

PCR products were each loaded in a volume of 5 μl into a 1% agarose gel,and electrophoresed, followed by identifying bands in a Gel-Doc.

In the mouse osteoblast cell line 3T3-L1 which was incubated with thepeptide of SEQ ID NO: 1, 3, or 5 for three days, decreased expressionlevels of the adipogenic marker aP2 were observed (FIGS. 3a-c ).

Also, when incubated for three days with concentrations of 0.1 μg/ml, 1μg/ml, and 10 μg/ml of a complex of peptides of SEQ IS Nos: 1, 3 and 7,the mouse osteoblast cell line was observed to decrease in theexpression of the adipogenic markers PPARγ, ACC, and aP2, like thepositive control cells treated with 100 ng/ml TNFα (FIG. 4).

1-3. Expression Observation of Adipogenesis and Lipolysis InducingProteins by Use of Pre-Adipocyte

3T3-L1 cells (pre-adipocytes) were seeded at a density of 3×105cells/well into 6-well plates. After 24 hours of culturing, the cellswere incubated for 14 days with predetermined concentrations (0.1, 1,and 10 μg/ml) of the peptide complex in a 37° C. incubator. Cell lysatesobtained by treatment with a cell lysis buffer were used for proteinquantitation, followed by Western blotting with an anti-PPARγ antibody(Santa Cruz Biotechnology, USA), which is an antibody against anadipogenic marker, and an anti-pHSL antibody (Santa Cruz Biotechnology,USA), which is an antibody against an lipolytic marker.

When treated with the peptide complex by concentration, the cells wereobserved to decrease in the expression of the adipogenic marker PPARγ ina dose-dependent manner while all increasing in the expression of thelipolysis marker pHSL (FIG. 5).

Example 2: Assay for Lipolytic Activity 2-1. Increased Expression ofGenes Involved in Lipolysis

3T3-L1 cells (pre-adipocytes) were seeded at a density of 3×105cells/well into 6-well plates. After 24 hours of culturing, the cellswere incubated for 14 days with predetermined concentrations (0.1, 1,and 10 μg/ml) of the peptides in a 37° C. incubator (positive control:100 ng/ml TNFα (SIGMA)). The cells were harvested and treated with anRNA extraction solution (Easy Blue, Intron) to prepare RNA from whichcDNA was then synthesized using an RT premix (Intron). PCR was performedusing primers for markers (AMPK-α1 and CGI58), and a PCR premix(Intron).

Target-specific primer sequences for PCR of lipolytic markers were asfollows: AMPK-α1 forward primer sequence, 5′-TGACCGGACATAAAGTGGCTGTGA-3′and AMPK-α1 reverse primer, 5′-TGATGATGTGAGGGTGCCTGAACA-3′(annealingtemperature, 60° C.); CGI58 forward primer sequence,5′-TGTGCAGGACTCTTACTTGGCAGT-3′ and CGI58 reverse primer,5′-GTTTCTTTGGGCAGACCGGTTTCT-3′(annealing temperature, 60° C.).

PCR products were each loaded in a volume of 5 μl into a 1% agarose gel,and electrophoresed, followed by identifying bands in a Gel-Doc.

In all of the pre-adipocytes (3T3-L1) which were incubated with thepeptides, increased expression levels of the lipolytic markers AMPK-α1and CGI-58 were detected (FIGS. 6a-c ). In addition, treatment with thepeptide complex was observed to increase the expression of AMPK-α1 andCGI-58 in dose-dependent manners and to higher levels compared to thepositive control TNFα 100 ng/ml treatment (FIG. 6d ).

2-2. Expression Observation of Lipolysis Inducing Proteins by Use ofPre-Adipocyte

3T3-L1 cells (pre-adipocytes) were seeded at a density of 3×105cells/well into 6-well plates. After 24 hours of culturing, the cellswere incubated for 14 days with predetermined concentrations (0.1, 1,and 10 μg/ml) of the peptide complex in a 37° C. incubator (positivecontrol: 100 ng/ml TNFα(SIGMA)). Cell lysates obtained by treatment witha cell lysis buffer were used for protein quantitation, followed byWestern blotting with an anti-ATGL antibody (Santa Cruz Biotechnology,USA), which is an antibody against an lipolytic marker.

The expression of the lipolytic marker ATGL was increased by treatmentwith the peptide complex (FIG. 7).

2-3. Fluorescence Microscopic Observation of Expression of LipolysisInducing Protein by Use of Pre-Adipocyte

3T3-L1 cells (pre-adipocytes) were seeded at a density of 3×105cells/well into 6-well plates. After 24 hours of culturing, the cellswere incubated for 14 days with the individual peptides or the peptidecomplex (1 μg/ml) in a 37° C. incubator (positive control: 100 ng/mlTNFα (SIGMA)). Thereafter, the cells were fixed with 70% ethanol andthen subjected to immunostaining with an anti-phospho-HSL antibody(Santa Cruz Biotechnology, USA) to observe the cellular expression ofphospho-HSL, a lipolytic marker.

From the experimental data, the peptides alone (FIGS. 8a-c ) and thepeptide complex (FIG. 8d ) were both observed to increase the expressionof the lipolytic marker phospho-HSL.

2-4. Quantitation of Lipolysis Product Glycerol

After being taken from the abdomens of obesity-induced mice, adiposetissues were plated at a density of 100 mg/well into 24-well cultureplates and cultured in a culture medium (1 ml Krebs-Ringer buffercontaining 25 mM HEPES, 5.5 mM glucose, and 2% (w/v) bovine serumalbumin). In this regard, the tissues were incubated for 48 hours with0.1 μg/ml, 1 μg/ml, and 10 μg/ml of the peptide complex whereas 100ng/ml of TNFα was used as a positive control. Glycerol produced duringlipolysis was quantitatively analyzed.

As is understood from the experimental data, the amount of glycerolresulting from lipolysis by treatment with the peptide complex wasincreased in a dose-dependent manner and greater than that produced upontreatment with the positive control TNFα (FIG. 9).

2-5. Lysis Effect on Adipose Tissue Isolated From Obese Mouse

Adipose tissues are divided into white fat and brown fat by color andinto subcutaneous fat, abdominal fat, mesentery fat (visceral fat), andepididymal fat by tissue. After body anatomization, lipoectomy wasperformed on the tissues. White fats were isolated, plated in an amountof 100 mg/well into 24-well plates, and then incubated for 72 hours withconcentrations of the peptide complex in a culture medium (1 mlKrebs-Ringer buffer containing 25 mM HEPES, 5.5 mM glucose, and 2% (w/v)bovine serum albumin). The fats were sectioned into slices which werethan dyed with hematoxylin and eosin. Sizes of adipocytes were comparedunder a microscope (TS100 Nikon) with 100× magnification.

Compared to the control, the fats treated with various concentrations ofthe peptides decreased in size (FIG. 10a ). In addition, when treatedwith the peptide complex, adipose tissues having distinct cell membranecompartments were observed in cell size, as measured by a program (FIG.10b ).

2-6. Observation of Lipolytic Marker in Adipose Tissue

An adipose tissue taken from the abdomen of an obesity-induced mouse wasplated in an amount of 100 mg per well into 24-well culture plates andincubated for 48 hours with the peptide complex while TNFα 100 ng/ml wasused as a positive control. The labeled lipolytic marker phospho-HSL(green fluorescent) was detected.

Treatment with the peptide complex was observed to increase theexpression level of the lipolytic marker phospho-HSL in adipose tissues(FIG. 11).

Example 3: Adipogenesis-Suppressive and Lipolysis-Promotive Effect inExperimental Animal Weight Loss and Adipogenesis Suppression in High-FatDiet-Fed Animal

Models DIO (diets induced obesity), which had become obese by feedinghigh-fat diets thereto, were used for anti-obesity experiments in whichTNFα 5 μg/ml was used as a positive control. For a control, a generaldiet, not a high-fat diet, was fed. In the experiment, a high-fat dietwas fed for 12 weeks while the peptide complex or the positive controlwas applied. During the experiment, the weight was monitored.

TNFα and the anti-obesity compounds were intraperitoneally injected atPM 3-4 o'clock every week for 12 weeks. Weights and meal sizes weremeasured just before the initial injection and then regularly atintervals of one week. Blood samples were taken from tail veins afterthe experiments of drug injection and measured for blood sugar levels,using Accu-Check Active (Roche) and analyzed for cholesterol levels,using Cholesterol calculation Kit (BioVision). Adipose tissues aredivided into white fat and brown fat by color and into subcutaneous fat,abdominal fat, mesentery fat (visceral fat), and epididymal fat bytissue. After lipoectomy, the fats thus obtained from the tissues wereobserved. For histological examination, the fats were fixed with 10%neutral buffered formalin, embedded in paraffin blocks, cut into 5μm-thick sections, and dyed with hematoxylin and eosin. To analyze thephosphorylation of the lipolytic marker HSL, fluorescent staining wascarried out with an anti-pHSL antibody. A tissue sample was made,mounted on glycerine jell mounting media, and covered with a coverglass. The tissues were observed under a microscope (Nikon, TS100), witha built-in digital camera taking images thereof.

Over 12 weeks from the initial stage to the final stage of theexperiment, mice were measured to increase in weight from 20.9 g to28.74 g when fed with a general diet and from 20.99 g to 49.5 g when fedwith a high-fat diet. In the mice fed with a high-fat diet with thepeptide complex injected thereto, the weight gain was reached only to36.76 g after 12 weeks from the initial weight of 21.1 g, indicating asignificant reduction of weight gain (174.2%), compared to the high-fatdiet-fed control (235.8%) (Table 2 and FIG. 12).

After completion of the 12-week experiment, in addition, the micetreated with the peptide complex, were observed to maintain their bodysizes in similar patterns to those of the normal mice (general diet),but not to those of the high-fat diet-fed mice, as analyzed on theimages (FIG. 13).

After 12 weeks of the experiment, the mice were subjected to micro-CT toexamine fat distribution across the body. As a result of the micro-CTdata of fats (yellow) in the body, the fat distributed across the bodywas remarkably increased in the high-fat diet-fed mice, compared to thegeneral diet-fed control while a significantly reduced level of fatsdistributed across the body was observed in the group which were treatedwith the peptide complex with the high-fat diet fed thereto (FIG. 14).

The mice which completed micro-CT imaging were anatomized to extract theadipose tissues distributed across the body. Volumes of the adiposetissues were compared. As a result, the fat extracted from the high-fatdiet-fed mice was greater than that from the general diet-fed mice, witha significant reduction in the fat volume in the mice treated with thepeptide complex plus the high-fat diet (FIG. 15).

Fats were isolated, and dyed with H&E to visualize fat sizes. Smallersizes of fats were observed in the mice treated with both the high-fatdiet and the peptide complex than in the high-fat diet-fed control (FIG.16a ). Fat size analysis through a program showed that, when the fatsize of the general diet-fed control was assumed to be 100%, a fat sizewas increased to 127% in the high-fat diet-fed group, but decreased to97% in the group treated with the high-fat diet and the peptide complex(FIG. 16b ).

The fats were isolated and examined for the expression level of thelipolytic marker phospho-HSL in adipose tissues. The mice treated withboth the high-fat diet and the peptide complex were observed to have anelevated expression level of phospho-HSL (FIG. 17).

Blood cholesterol levels in the mice after the experiment were measured.As a result, the blood cholesterol level was 2.52 μg/ml in the generaldiet-fed mice, 3.5 μg/ml in the high-fat diet-fed mice, and 2.86 μg/mlin the mice treated with both the high-fat diet and the peptide complex,indicating that the peptide complex reduced the cholesterol level thatelevated with obesity (FIG. 18).

Blood sugar levels after completion of the experiment were 174 mg/dL inthe general diet-fed mice, and increased to 235 mg/dL in the high-fatdiet-fed mice. However, a blood sugar level of 183 mg/dL was measured inthe mice treated with both the high-fat diet and the peptide complex,with a significant reduction therein (FIG. 19).

Example 4: Blood Sugar Control Effect on Blood Sugar Control

In this animal experiment, C57BL/6 (normal mouse) (purchased fromSamtako Inc.) and female C57BLKS/JLepr (diabetes model mouse, db/dbmouse) (purchased from Central Lab. Animal Inc.) were used, togetherwith the peptide complex as an anti-diabetes and/or anti-obesityeffective material, and sitagliptin as a positive control drug. In thisExample, the anti-diabetes and/or anti-obesity effective complex wasevaluated for acute anti-diabetes efficacy (single administration) in anormal mouse model and a genetically potential-diabetic model, using GTT(glucose tolerance test), which is a representative diagnostic methodfor diabetes. Mice were bred per cage at a temperature of 22-24° C. anda relative humidity of 50-30%, with four per cage. The mice was under150-300 Lux light from AM 8 o'clock to PM 8 o'clock with 12 light/12dark cycles. They were given free access to a general diet (18% protein,manufactured in 2018, Harlan Laboratories Inc, USA). To begin with, themice were starved for 4 hours or longer before ITT experiment and for 12hours before GTT experiment. The complex was orally administered byforce with the aid of a disposable oral administration syringe one hourbefore GTT experiment. For GTT experiment, the mice were allowed tofreely access to a high-fat diet on 0 (zero) hour after experimentstarted. After 40 min of free access to a high-fat diet, blood samplesfor use in examining blood glucose levels were taken from the tail veinat intervals of 0, 30, 60, 90, 120, and 180 min. Blood glucose levelswere measured using Accu-Chek active (Roche). Sitagliptin, used as atherapeutic agent for diabetes, was selected as a positive control drug,and administered at a dose of 100 mg/kg. The complex selected as ananti-diabetes and/or anti-obesity effective candidate was administeredat a dose of 100 mg/kg to experimental groups of four mice.

As a result, the peptide complex exhibited a reductive effect on bloodsugar levels in which the blood sugar level increased by the high-fatdiet was reduced by treatment with the peptide complex. In thediabetes-induced mouse models, the high blood sugar level was decreasedby the complex (FIGS. 20a and 20b ). Further, lower blood cholesterollevels were detected in the group treated with both the high-fat dietand the peptide complex than the high-fat diet-fed control (FIG. 21).

In addition, after starvation for 16 hours, DB/DB diabetes-induced micewere fed for 30 min and then administered with the peptides. Blood sugarlevels were measured over times.

The blood sugar levels in the groups respectively treated with thepeptides of SEQ ID NOS: 1, 3, and 5 were observed to decrease in atime-dependent manner (FIGS. 22a-22c ).

Example 5: Promotion of Expression of Insulin and Insulin-Like GrowthFactor Promotion of Expression of Insulin and Insulin-Like Growth Factor

3T3-L1 cells (pre-adipocytes) were seeded at a density of 3×105cells/well into 6-well plates and grown for 24 hours. Subsequently, thecells were incubated with various concentrations (10 ng-1 μg/ml) of thepeptides for 14 days in a 37° C. incubator. Proteins were extracted fromcell lysates which were obtained by treatment with cell lysis buffer,quantitatively analyzed, and subjected to Western blotting using ananti-IGF-1 antibody, which is an antibody against the lipolytic marker,and an insulin antibody (Santa Cruz Biotechnology, USA).

From the data, it was observed that the peptide of SEQ ID NO: 7increased the expression of IGF-1 and insulin in dose-dependent manners(FIG. 23).

Example 6: Observation of Blood Sugar Level Reducing Effect in ClinicalExperiment Reduction of Blood Sugar Level by Intake of the Complex

A brief clinical test was performed on persons 45-65 years old who had afasting blood glucose level of 170 mg/dL or higher. They were ingestedwith a complex formulation 30 min after meals. Blood samples were takenat intervals of 30, 60, 90, 120, 150, and 180 min from the persons, andmeasured for glucose level, using Accu-Chek active (Roche).

A reduction of blood sugar level by the complex formulation was observedin all the tested persons (FIGS. 25a-25d ).

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

1. A peptide having one selected from the group consisting of the amino acid sequences of SEQ ID NOS: 1 to
 7. 2. A peptide, having one selected from the group consisting of the amino acid sequences of SEQ ID NOS: 1 to 7 and exhibiting anti-obesity or anti-diabetes activity.
 3. The peptide of claim 2, wherein the peptide suppresses adipogenesis.
 4. The peptide of claim 2, wherein the peptide reduces expression of PPARγ (peroxisome proliferator-activated receptor gamma), ACC (acetyl-CoA carboxylase), or aP2 (adipose-specific fatty acid-binding protein 2).
 5. The peptide of claim 2, wherein the peptide promotes lipolysis.
 6. The peptide of claim 2, wherein the peptide increases expression of pHSL (phospho-hormone-sensitive lipase), AMPK-α1 (AMP-activated protein kinase α1), CGI-58 (comparative gene identification-58), or ATGL (adipose triglyceride lipase).
 7. The peptide of claim 2, wherein the peptide reduces a blood sugar level.
 8. A peptide complex, exhibiting anti-obesity or anti-diabetes activity and including the following peptide combination: (a) a peptide of SEQ ID NO: 1; (b) a peptide of SEQ ID NO: 3; and (c) a peptide of SEQ ID NO: 6 or
 7. 9. The peptide complex of claim 8, wherein the peptide complex reduces sizes of adipocytes.
 10. The peptide complex of claim 8, wherein the peptide complex reduces a blood cholesterol level.
 11. A pharmaceutical composition, comprising the peptide of claim 2 as an effective ingredient for preventing or treating obesity.
 12. A pharmaceutical composition, comprising the peptide complex of claim 8 as an effective ingredient for preventing or treating obesity.
 13. A pharmaceutical composition, comprising the peptide of claim 2 as an effective ingredient for preventing or treating diabetes.
 14. A pharmaceutical composition, comprising the peptide complex of claim 8 as an effective ingredient for preventing or treating diabetes.
 15. A method for preventing or treating obesity or diabetes, comprising administering a pharmaceutically effective amount of the peptide of claim 2 into a subject.
 16. A method for preventing or treating obesity or diabetes, comprising administering a pharmaceutically effective amount of the peptide complex of claim 8 into a subject. 